Download Toll-like receptors and immune regulation: their direct and indirect

IMMUNOLOGY
REVIEW ARTICLE
Toll-like receptors and immune regulation: their direct and indirect
modulation on regulatory CD4+ CD25+ T cells
Guangwei Liu and Yong Zhao*
Summary
Transplantation Biology Research Division,
State Key Laboratory of Biomembrane and
Membrane Biotechnology, Institute of Zoology,
Chinese Academy of Sciences, Beijing, China
Regulatory CD4+ CD25+ T (Treg) cells with the ability to suppress host
immune responses against self- or non-self antigens play important roles
in the processes of autoimmunity, transplant rejection, infectious diseases
and cancers. The proper regulation of CD4+ CD25+ Treg cells is thus critical for optimal immune responses. Toll-like receptor (TLR)-mediated
recognition of specific structures of invading pathogens initiates innate as
well as adaptive immune responses via antigen-presenting cells (APCs).
Interestingly, new evidence suggests that TLR signalling may directly or
indirectly regulate the immunosuppressive function of CD4+ CD25+ Treg
cells in immune responses. TLR signalling may shift the balance between
CD4+ T-helper cells and Treg cells, and subsequently influence the outcome of the immune response. This immunomodulation pathway may
therefore have potential applications in the treatment of graft rejection,
autoimmune diseases, infection diseases and cancers.
doi:10.1111/j.1365-2567.2007.02651.x
Received 26 November 2006; revised
18 March 2007; accepted 3 May 2007.
Correspondence: Dr Yong Zhao,
Transplantation Biology Research Division,
State Key Laboratory of Biomembrane and
Membrane Biotechnology, Institute of
Zoology, Chinese Academy of Sciences,
Datun Road, Beijing, China 100101.
Email: [email protected]
Senior author: Yong Zhao
Keywords: Toll-like receptors; regulatory CD4+ CD25+ T cells; immune
response; immune tolerance; autoimmune disease
Introduction
The family of Toll-like receptors (TLRs) is a major class
of receptors that recognize molecular patterns associated
with pathogens including bacteria, viruses, fungi and protozoa. It was commonly accepted that TLR-mediated
recognition of specific structures of invading pathogens
initiates innate as well as adaptive immune responses via
dendritic cells (DCs) or other antigen-presenting cells
(APCs).1–3 However, there is emerging evidence that TLR
signalling participates in inflammation and immune
responses that are driven by self-, allo- or xenoantigens.4–7 TLR signalling has been demonstrated to be
involved in the immune recognition of allo- or xenografts
and the occurrence of autoimmunity both in experimental and in clinical studies.5,8–10 This observation was
strongly supported by the expression of TLRs on almost
all immune cells and the endogenous expression of their
ligands on mammalian cells.
However, there has recently been an explosion of
renewed interest in regulatory T (Treg) cells, especially
on three postulated CD4+ Treg cell populations: the naturally occurring and inducing CD4+ CD25+ Treg cells,
and two inducible populations, type 1 regulatory T (Tr1)
cells and type 3 regulatory T (Th3) cells.11–13 It is clear
now that the CD4+ CD25+ Treg cell population not only
critically contributes to the maintenance of self-tolerance
but also has the potential to prevent the immune rejection of allografts.14,15 Recent studies have shown that
TLR signalling may directly or indirectly regulate the
immunosuppressive function of CD4+ CD25+ Treg cells
in graft rejection, autoimmune diseases, infectious diseases and cancers,16–19 which is the focus of the present
review.
Abbreviations: APC, antigen-presenting cell; DC, dendritic cell; DD, death domain; Foxp3, Forkhead box protein 3; IFN,
interferon; IRAK, IL-1R-associated kinase; IRF-3, interferon regulatory factor 3; IL, interleukin; LPS, lipopolysaccharide; LTA,
lipoteichoic acid; MyD88, myeloid differentiation primary response protein 88; Mal, MyD88-adaptor-like; NF-jB, nuclear factor
jB; PAMP, pathogen-associated molecular pattern; TGF, transforming growth factor; TNF, tumour necrosis factor; Treg cell,
regulatory T cell; Tr1 cell, type 1 regulatory T cell; TRAF6, tumour necrosis factor receptor-associated factor 6; TRAM,
TRIF-related adaptor molecule; TIRAP, TIR-associated protein; TRIF, TIR domain containing adaptor inducing IFN-b; Th3 cell,
type 3 regulatory T cell; TLR, Toll-like receptor.
Ó 2007 Blackwell Publishing Ltd, Immunology, 122, 149–156
149
Guangwei Liu and Yong Zhao
TLRs and their ligands
TLRs have recently emerged as a critical component of
the innate immune system for detecting microbial infections, APC activation and the induction of adaptive
immune responses.20,21 At least 13 TLRs have been
identified in humans and mice to date.20,22–24 The initial
role of TLRs in innate immunity against micro-organisms
has been studied extensively.25–31 They recognize limited but highly conserved molecular structures, socalled pathogen-associated molecular patterns (PAMPs)
(Table 1).22,32–34 Thus, TLRs recognize conserved molecular features of bacteria, fungi and viruses.
Interestingly and importantly, some endogenous ligands
of TLRs were identified recently. Heat shock proteins
(hsps) such as hsp60, hsp70, hsp90 and glycoprotein 96
(gp96; the endoplasmic reticulum form of hsp90) of bacterial and mammalian origins have been shown to induce
the inflammatory response.35 Using macrophages from
C3H/HeJ mice with a TLR4 gene point mutation,
researchers demonstrated that the effects of recombinant
human hsp60 (rhhsp60) were dependent on TLR4, suggesting that hsp60 might be an endogenous TLR4 ligand.22,30,36 Since then, using TLR4 mutant mice, TLR2
knockout mice, and/or fibroblasts transfected with TLR2,
TLR3 or TLR4 cDNA, researchers have found that fibrinogen, surfactant protein A, fibronectin extra domain
A, heparan sulphate, soluble hyaluronan and murine
b-defensin 2 are endogenous ligands for TLR4; hsp60,
hsp70, gp96 and high mobility group 1 (HMGB1) protein
are endogenous ligands for both TLR2 and TLR4;32,34,35,37
and mRNA is an endogenous ligand for TLR338
(Table 1).
The identification of endogenous ligands for TLRs
raised the possibility that TLRs may be involved in selftolerance and surveillance, which are critical for the
occurrence of autoimmune diseases and cancers.3,39 TLRs
recognize not only the endogenous and exogenous molecules but also the degradation products of endogenous
macromolecules such as heparan sulphate and polysaccharide fragments of hyaluronan, which indicate tissue
injury, infection and/or tissue remodelling.22,32–34,36,37
However, fibrinogen is normally present in the circulation
at high concentrations.35,37 Thus, monocytes, DCs and
sinusoidal macrophages such as hepatic Kupffer cells and
splenic macrophages are constantly exposed to fibrinogen.
However, surfactant protein A is normally present in lung
alveoli where alveolar macrophages reside.39,40 Life-long
continuous exposure of immune cells to these TLR ligands may be potentially deleterious to the host. However,
the question of why the recognition of endogenous molecules, such as fibrinogen and surfactant protein A, by
TLR4 does not mediate the activation of APCs in a physical situation needs to be addressed.
Table 1. Toll-like receptors (TLRs) and their exogenous and endogenous ligands
TLR
Exogenous ligands
Endogenous ligands
References
TLR1
TLR2
Bacterial triacyl lipopeptides and proteins in parasites
Bacterial diacyl lipopeptides, lipoteichoic acid from
Gram-positive bacteria, and zymosan from the cell wall
of yeast
Double-stranded RNA from viruses
Endotoxin (LPS) from Gram- negative bacteria
Unknown
hsp70, gp96, necrotic cells
and HMGB1
22
22,29,30,32,34
mRNA
hsp60, hsp70, hyaluronan, lung
surfactant protein A, fibronectin,
fibrinogen, heparan, HMGB1 and
b-defensin 2
Unknown
Unknown
Unknown
Unknown
Chromatin–IgG complexes
Unknown
Unknown
20
22,30,31,34
41
20
25
52
26
17
22,27
Unknown
Unknown
22,27
20
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
TLR10
TLR11
TLR12
TLR13
Flagellin from mobile bacteria
Partnered with TLR2
Single-stranded RNA from viruses
Same as TLR7
CpG DNA from bacteria or viruses
Unknown
Profilin, a protein from the protozoan pathogen
Toxoplasmosis gondii that can cause miscarriage; may
also respond to components of bacteria that cause
bladder and kidney infections
Unknown
Unknown
gp96, glycoprotein 96 (the endoplamic reticulum form of hsp90); HMGB1, high- mobility group box 1; hsp, heat shock protein; IgG, immunoglobulin G; LPS, lipopolysaccharide.
150
Ó 2007 Blackwell Publishing Ltd, Immunology, 122, 149–156
Toll-like receptor modulation on regulatory CD4+ CD25+ T cells
TLR expression on T cells
TLR expression has been detected in many types of
immune cells, including different subsets of DCs, T cells,
neutrophils, eosinophils, mast cells, macrophages, monocytes and epithelial cells (Table 2).22,41,42 Importantly, the
expression of TLRs is related to the functional states of
different subtypes of T cells. Studies have shown that
naı̈ve CD4+ T cells do not express significant levels of
TLR2 and TLR4 mRNA and intracellular proteins. Only a
few CD3+ T cells express TLR1, TLR2 or TLR4 on the cell
surface when they have not been activated.43 However,
activated/memory T cells express appreciable levels of cell
surface TLR2 and TLR4.5,21,44 T-cell receptor (TCR) stimulation by cross-linked anti-CD3 monoclonal antibody
(mAb) induces cell surface expression of TLR2 and TLR4
on naı̈ve human and murine CD4+ T cells.5,45 By contrast, TCR stimulation significantly down-modulates
surface TLR5 expression on human CD4+ T cells.41
Moreover, TLR3, TLR6, TLR7 and TLR9 are also
expressed on CD4+ T cells.46 Thus, some TLRs may function as costimulator receptors for antigen-specific T-cell
development and immune responses, and participate in
the maintenance of T-cell memory.47,48 These data indicate that pathogens, via their PAMPs, may contribute
directly to the perpetuation and activation of memory
T cells.
Some TLRs are expressed on CD4+ CD25+ Treg cells
(see Table 4 below). It has been reported that
CD4+ CD25+ Treg cells in naı̈ve mice selectively express
TLR4, TLR5, TLR7 and TLR8, whereas TLR1, TLR2,
TLR3 and TLR6 appear to be more widely expressed on
CD4+ T cells, and not confined to CD4+ CD25+ Treg
Table 2. The expression pattern of Toll-like receptors (TLRs) on
different immune cells
TLR
Expression on immune cells
References
TLR1
TLR2
TLR3
TLR4
TLR5
TLR6
Most cell types including DCs and B cells
PMLs, DCs, monocytes and T cells
DCs, NK cells and T cells
Macrophages, DCs and T cells
Monocytes, DCs, NK cells and T cells
High expression in B cells and DCs;
low in monocytes and NK cells
B cells, DCs, monocytes and T cells
Monocytes, DCs; low in NK and T cells
DCs, B cells, PMLs, macrophages,
NK cells and microglial cells
B cells; low in DCs
Unknown
Unknown
Unknown
22
21,29,30
20,38
20,30,31
20,41
20,63
TLR7
TLR8
TLR9
TLR10
TLR11
TLR12
TLR13
25,38
26,52
22,26
17,38
22,27
22,27
20
DC, dendritic cell; NK, natural killer; PML, peripheral mononuclear
leucocyte.
Ó 2007 Blackwell Publishing Ltd, Immunology, 122, 149–156
cells.49,50 The distinct expression patterns of TLRs on
CD4+ CD25+ Treg cells support the potential involvement
of these TLRs in the regulation of CD4+ CD25+ Treg
cells.
TLRs, CD4+ CD25+ Treg cells and the immune
response
Naturally occurring and antigen-induced CD4+ CD25+
Treg cells have been extensively studied in mice and
humans. Depletion of the naturally occurring subset of
CD4+ CD25+ Treg cells results in various types of autoimmune disease.11,12,27,41 CD4+ CD25+ Treg cells inhibit a
wide range of autoimmune and inflammatory manifestations such asgastritis, oophoritis, orchitis, thyroiditis,
inflammatory bowel disease and spontaneous autoimmune diabetes.11,12,27,41,51 Although CD4+ CD25+ Treg
cells play a critical role in the regulation of immunity, we
know little about the regulation of CD4+ CD25+ Treg
cells. Interestingly, new evidence suggests that TLR signalling may directly or indirectly regulate the immunosuppressive function of CD4+ CD25+ Treg cells in immune
responses.16,17
To date, differential TLR expression on CD4+ T cells
and CD4+ CD25+ Treg cells has been found, suggesting
that TLRs may be directly involved in adaptive immune
responses, but how TLRs directly regulate adaptive
immune responses is still not fully understood.52,53
Recently, a close relationship between TLRs and autoimmune diseases has been reported in mouse models. In
a mouse model of systemic lupus erythematosus (SLE),
when TLR9 was absent, SLE was exacerbated with
increased activation of lymphocytes and plasmacytoid
DCs as well as enhanced levels of immunoglobulin G
(IgG) and interferon-a (IFN-a) in sera. In contrast,
TLR7-deficient mice had ameliorated SLE with decreased
lymphocyte activation and serum IgG levels.54 These data
reveal opposing inflammatory and regulatory roles for
TLR7 and TLR9 in SLE. Importantly, it is interesting that,
in in vitro CD4+ CD25+ Treg cell suppression assays,
engagement of TLR4 or TLR9 on freshly isolated mouse
splenic DCs could significantly abrogate the immunosuppressive function of CD4+ CD25+ Treg cells, by rendering
effector T cells resistant to CD4+ CD25+ Treg cell-mediated suppression through DC expression of interleukin-6
(IL-6), but not the effects of costimulation.39 Thus, the
regulation of TLRs on CD4+ CD25+ Treg cells may
directly or indirectly play a role in the occurrence of
autoimmune diseases.
There is accumulating evidence of significant alterations
in TLR expression on T cells in patients who have infectious diseases, although it is unclear whether altered TLR
expression on T cells contributes directly to pathophysiology or immune defence in these patients.55,56 Furthermore, cross-talk between TLRs and CD4+ CD25+ Treg
151
Guangwei Liu and Yong Zhao
Table 3. Potential roles of Toll-like receptors (TLRs) as therapeutic
targets in inflammatory diseases, cancers and autoimmune diseases
TLR
Related diseases
References
TLR1
Bacterial/fungal diseases,
Gram-positive sepsis
Bacterial/fungal diseases,
Gram-positive sepsis
Viral diseases
Bacterial diseases, Gram-negative
sepsis, chronic inflammation,
autoimmune diseases, cancers,
atherosclerosis
Bacterial diseases
Mycobacterial diseases
Viral diseases
Viral diseases
Bacterial and viral diseases,
autoimmune diseases, cancers
Unknown
Unknown
Unknown
Unknown
20,22
TLR2
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
TLR10
TLR11
TLR12
TLR13
21,29,30
10,20
20,30
41
3,20
20,38
20,52
26
27
22,27
22,27
20,52
cells in tumours has also been noted.5,21 Elucidation of
TLR signalling in adaptive immune responses in terms of
both direct and indirect regulation of the immunosuppressive function of CD4+ CD25+ Treg cells may therefore have therapeutic benefits in inflammation, cancers
and autoimmune diseases (Table 3).
The direct regulatory effects of TLRs on CD4+ CD25+
Treg cells
TLR2–/– mice, unlike TLR4–/– mice, contain significantly
fewer CD4+ CD25+ Treg cells than control mice.21
Administration of TLR2 ligands to wild-type mice
results in significantly increased CD4+ CD25+ Treg cell
numbers.5,21 In the presence of a TLR2 agonist, such
as the synthetic bacterial lipoprotein Pam3Cys-SK4,
CD4+ CD25+ Treg cells expand markedly but their
immunosuppressive function is temporarily abrogated.5
However, studies have shown that hsp60 could act as a
costimulator of CD4+ CD25+ Treg cells.21,46,57 Treatment
of CD4+ CD25+ Treg cells with hsp60 or its peptide p277
before anti-CD3 mAb-induced activation significantly
enhanced the ability of the CD4+ CD25+ Treg cells to
down-regulate the function of CD4+CD25– or CD8+ target T cells, detected through the inhibition of target T-cell
proliferation and IFN-c and tumour necrosis factor
(TNF)-a secretion.58 The enhanced costimulatory effects
of hsp60 on CD4+ CD25+ Treg cells involved innate signalling via TLR2, which led to activation of protein
kinase C, phosphatidyl-inositol-3-kinase, and p38.58 hsp60treated CD4+ CD25+ Treg cells suppressed target T cells
both by cell contact mechanisms and by secretion of cytokine transforming growth factor (TGF)-b and IL-10.58
Thus, hsp60, a self-molecule, can down-regulate adaptive
immune responses by up-regulating CD4+ CD25+ Treg
cells innately through TLR2 signalling and not through
APCs. Thus, the effect of TLR2 on CD4+ CD25+ Treg
cells is somewhat controversial and further investigation
is required.
Researchers observed that exposure of CD4+ CD25+
Treg cells to the TLR4 ligand lipopolysaccharide (LPS)
induced up-regulation of several activation markers and
enhanced their survival or proliferation.49 The proliferative response does not require APCs and is augmented by
TCR triggering and IL-2 stimulation.49 Most importantly,
LPS treatment increases the immunosuppressive ability of
CD4+ CD25+ Treg cells by 10-fold.49 Moreover, LPSactivated CD4+ CD25+ Treg cells can efficiently control
the occurrence of naı̈ve CD4+ effector T cell-mediated
diseases.49 These findings provide the first evidence that
Table 4. Toll-like receptor (TLR) expression
and function on CD4+ CD25+ Treg cells
Effects on CD4+ CD25+ Treg cells
TLR
Effector
T cells
CD4+CD25+
Treg cells
Cell
proliferation
TLR1
TLR2
+
+
+
+
?
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
+
+
+
+
+
+
++
++
+
++
++
+
Yes
No
Yes
Suppression
Up-regulation/
down-regulation (?)
Up-regulation
Up-regulation
Down-regulation
Blocking (partially
via effector T cells)
References
49
21,58
49
41,60
49
49
48
65
+, normal expression; ++ higher expression compared with effector T cells.
152
Ó 2007 Blackwell Publishing Ltd, Immunology, 122, 149–156
Toll-like receptor modulation on regulatory CD4+ CD25+ T cells
CD4+ CD25+ Treg cells respond directly to pro-inflammatory bacterial products, a mechanism that is likely to
contribute to the control of inflammatory responses.
However, others failed to observe effects of LPS on
CD4+ CD25+ Treg cells.41,45,59 Thus, LPS-induced signalling on CD4+ CD25+ Treg cells is still controversial.
TLR5 ligand flagellin has important effects in regulating
mucosal immune responses.41,60,61 Both human CD4+
CD25+ Treg cells and CD4+CD25– T cells express TLR5
at levels comparable to those on monocytes and DCs.
Costimulation of CD4+ effector T cells with antiCD3 mAb and flagellin resulted in enhanced proliferation
and production of IL-2 at levels equivalent to those
achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+ CD25+ Treg cells, but rather potently
increased their immunosuppressive capacity and enhanced
expression of Forkhead box protein 3 (Foxp3).41,60 However, in a tumour mouse model, administration of flagellin, which is specifically recognized by TLR5, at 8–10 days
after tumour implantation produced significant inhibition
of the growth of the antigenic tumour, which was associated with an increased IFN-c:IL-4 ratio and a decreased
frequency of CD4+ CD25+ Treg cells.62 In contrast, flagellin administered at the time of tumour implantation
led to accelerated tumour growth, which was associated
with a decreased IFN-c:IL-4 ratio and an increased
CD4+ CD25+ Treg cell frequency.62 Whether the contrasting effects of activation of TLR5 by flagellin on tumour
growth are attributable to the different impacts on effector T cells and CD4+ CD25+ Treg cells and/or the different effects on naı̈ve and activated/memory CD4+ CD25+
Treg cells has not yet been determined.
In addition, TLR8 could directly reverse the immunosuppressive function of CD4+ CD25+ Treg cells.21,52 It has
been reported that CpG-A and poly (G10) oligonucleotides could directly reverse the immunosuppressive function of CD4+ CD25+ Treg cells in the absence of DCs, but
the exact functional ingredients were not identified in that
study.49 Further experiments indicated that short poly (G)
oligonucleotides [poly (G2), poly (G3) and poly (G4) with
phosphorothioate linkages] had a more potent ability to
reverse CD4+ CD25+ Treg cell function than longer oligonucleotides [poly (G5), poly (G7) and poly (G10)].52
Interestingly, when TLR8 and myeloid differentiation primary response protein 88 (MyD88) were knocked down
using an RNA interference method, the response ability of
CD4+ CD25+ Treg cells to poly (G) oligonucleotides was
abolished. Consistent with these results, TLR8 was consistently expressed by naturally occurring as well as inducing CD4+ CD25+ Treg cells.52,63,64 A recent study more
clearly demonstrated that the immunosuppression of
CD4+ CD25+ Treg cells is abrogated by TLR8 triggering
directly on the CD4+ CD25+ Treg cells, not on the CD4+
effector T cells.52 These results consistently support the
Ó 2007 Blackwell Publishing Ltd, Immunology, 122, 149–156
hypothesis that the TLR8–MyD88 signalling pathway
directly controls the immunosuppressive function of CD4+
CD25+ Treg cells without the involvement of APCs.
The TLR9 ligand CpG oligodeoxynucleotide synergizes
with anti-CD3 mAb to induce proliferation of both rat
CD4+ CD25– and CD4+ CD25+ Treg cells.65 Surprisingly,
TLR9 ligand partially abrogates the suppressive activity
mediated by CD4+ CD25+ Treg cells, which is partially
attributable to the direct effect of TLR9 ligand on effector
T cells which are rendered more resistant to the regulation exerted by CD4+ CD25+ Treg cells.65 Thus, TLR9 ligand may rapidly increase the host’s adaptive immunity by
expanding effector T cells and also by attenuating the
suppressive activity mediated by CD4+ CD25+ Treg cells.
Taking these results together, it is obvious that
CD4+ CD25+ Treg cells express certain TLRs. In general,
TLR2(?), TLR8 or TLR9 ligation abrogates or reverses the
immunosuppressive function of CD4+ CD25+ Treg cells,
whereas TLR2(?), TLR4 or TLR5 ligation enhances
CD4+ CD25+ Treg cell suppressive capacity (Table 4).
The mechanisms by which TLRs modulate the immunosuppressive ability of CD4+ CD25+ Treg cells are not
clear. One explanation is that the up-regulation or downregulation of Foxp3 expression following stimulation by
different TLRs may be related to the functional alteration
of CD4+ CD25+ Treg cells. However, how TLR signalling
affects Foxp3 expression needs to be addressed. Another
possibility to explain the abrogated immunosuppressive
function but enhanced proliferative capacity of CD4+
CD25+ Treg cells after TLR stimulation is suggested by
reports indicating that CD4+ CD25+ Treg cells rapidly
lose their ability to inhibit proliferation after receiving
strong activation signals. Experimental data support
the idea that TLR2 on murine CD4+ CD25+ Treg cells
might function as a strong costimulatory trigger.5,37 We
may speculate that some TLR-mediated signals force
CD4+ CD25+ Treg cells into the proliferative pathway,
which might be paralleled by the reversal of their immunosuppressive capabilities. However, TLR4 may induce
CD4+ CD25+ Treg cell proliferation while enhancing the
immunosuppressive ability of CD4+ CD25+ Treg cells,
which does not support this speculation.
The indirect regulatory effects of TLRs
on CD4+ CD25+ Treg cells
A central function of TLRs is to directly activate acute
antimicrobial defence systems. TLRs are known to induce
the production of antimicrobial proteins and peptides by
various cell types, including cells of myeloid origin in the
gut epithelium.4,41,66 Activation of resident macrophages
through TLRs also leads to production of various cytokines (IL-1, IL-6, TNF-a, etc.) and chemokines (monocyte
chemotactic protein 1, etc.), which collectively orchestrate
the acute inflammatory response to infections.53,67
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Guangwei Liu and Yong Zhao
DCs are pivotally positioned at the interface of innate
and adaptive immunity. Pathogen recognition is mediated
by TLRs which are expressed at high levels on the surface
of DCs. Analysis of MyD88-deficient mice demonstrated
the critical role of TLRs in DC maturation and induction
of adaptive immune responses.3,68,69 MyD88-deficient
mouse DCs could not prime antigen-specific naı̈ve T cells
in vitro.70 While antigen mixed with complete Freund’s
adjuvant (CFA), which contains several ligands for TLRs,
leads to a robust immune response in wild-type mice, it
fails to trigger T-cell proliferation and IFN-c production
in MyD88-deficient mice.71,72 These data suggest that
engagement of TLRs on DCs in vivo is required for DC
maturation, secretion of cytokines and chemokines and
antigen-presenting ability, which subsequently control the
choice of the effector T cells of adaptive immune
responses. TLR signalling in macrophages and DCs leads
to secretion of IL-12 which skews the resultant CD4+
effector T-cell response towards the T helper type 1 (Th1)
phenotype.23 Moreover, TLR triggering in APCs contributes to the adaptive immune response by indirectly controlling the immunosuppression of CD4+ CD25+ Treg
cells.53
Some researchers found that the immunosuppressive
function of CD4+ CD25+ Treg cells is critically dependent
on immature DCs and is readily reversed by TLR-induced
activation of DCs.59 The potential responsiveness of
CD4+ CD25+ Treg cells to IL-2 was increased by the
co-operative effects of IL-6 and IL-1, both of which are
produced by TLR-activated mature DCs.5,58,73 The proinflammatory cytokines produced by TLR-activated
mature DCs are required for reversal of CD4+ CD25+
Treg cell anergy, but whether they are required to overcome the immunosuppression of CD4+ CD25+ Treg
cells is a matter of controversy.39,59 However, cytokines
secreted by APCs in response to TLR ligands are also
important for CD4+ effector T cells to overcome
the immunosuppressive effects of CD4+ CD25+ Treg
cells.18,23,74
Some studies showed that decreased immunosuppression of CD4+ CD25+ Treg cells on effector T cells was
mediated by soluble factors produced by DCs in response
to the TLR4 ligand LPS or the TLR9 ligand CpG oligonucleotide.5,19,23 The activity of LPS-treated DC supernatants was specifically abrogated by neutralizing
anti-IL-6 mAbs, thus indicating the significance of IL-6 in
the control of CD4+ CD25+ Treg cells on CD4+ CD25–
effector T cells.19 These conclusions were further supported by the observation that the supernatant of LPS-stimulated DCs from IL-6–/– knock-out mice was unable to
reverse suppression of CD4+ CD25+ Treg cells.75 However, recombinant IL-6 did not substitute for DC supernatant. This suggests that, in addition to IL-6, some
factors, as yet undefined, are essential in this process.
However, preincubation of CD4+ CD25+ Treg cells with
154
the supernatant of LPS-activated DCs did not block the
immunosuppressive capacity of CD4+ CD25+ Treg cells,76
suggesting that the supernatant of LPS-activated DCs may
render CD4+ CD25– effector T cells refractory to suppression rather than blocking the immunosuppressive capacity
of CD4+ CD25+ Treg cells.23,59,76,77 This regulating network may allow CD4+ CD25+ Treg cells to maintain their
function and prevent self-reactive T-cell activation during
an ongoing immune response to infections and other
damage stimuli.
Although some TLR signalling on DCs by CpG or LPS
rendered CD4+ effector T cells refractory to CD4+ CD25+
Treg cell-mediated suppression, TLR-induced mature DCs
are also capable of stimulating the proliferation of
CD4+ CD25+ Treg cells,78,79 which may be beneficial for
the efficient down-regulation of the immune response as
soon as the pathogens have been cleared.
Perspectives and closing remarks
Recent studies have demonstrated that the immunosuppressive function of CD4+ CD25+ Treg cells can be regulated through TLR signalling. Various pathways, including
the indirect route via APCs and their cytokine products
as well as the direct effects of TLRs on CD4+ CD25+ Treg
cells, may collectively contribute to the generation, expansion and function of CD4+ CD25+ Treg cells. Further
studies are needed to clarify the molecular mechanisms
for the regulation of CD4+ CD25+ Treg cells via TLRs.
Acknowledgements
The authors wish to thank Mr Aqeel Javeed for his review
of the manuscript. This work was supported by grants
from the National Natural Science Foundation for Key
Programs (C30630060 to YZ), the National Natural Science Foundation for Young Scientists (C30600567 to GL),
the National Natural Science Foundation for Distinguished
Young Scholars (C03020504 to YZ), the National Basic
Research Program (973 Program, 2003CB515501, to YZ),
100 Quality Vocational Colleges of the Chinese Academy
of Sciences (2003-85 to YZ), and the Scientific Research
Foundation for Returned Overseas Chinese Scholars of the
State Education Ministry (2005-546 to YZ).
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